Htt Expression Research Articles

Personalized gene silencing therapeutics for Huntington disease

Gene silencing offers a novel therapeutic strategy for dominant genetic disorders. In specific diseases, selective silencing of only one copy of a gene may be advantageous over non-selective silencing of both copies. Huntington disease (HD) is an autosomal dominant disorder caused by an expanded CAG trinucleotide repeat in the Huntingtin gene (HTT). Silencing both expanded and normal copies of HTT may be therapeutically beneficial, but preservation of normal HTT expression is preferred. Allele-specific methods can selectively silence the mutant HTT transcript by targeting either the expanded CAG repeat or single nucleotide polymorphisms (SNPs) in linkage disequilibrium with the expansion. Both approaches require personalized treatment strategies based on patient genotypes. We compare the prospect of safe treatment of HD by CAG- and SNP-specific silencing approaches and review HD population genetics used to guide target identification in the patient population. Clinical implementation of allele-specific HTT silencing faces challenges common to personalized genetic medicine, requiring novel solutions from clinical scientists and regulatory authorities.

Silencing Mutant Huntingtin by Adeno-Associated Virus-Mediated RNA Interference Ameliorates Disease Manifestations in the YAC128 Mouse Model of Huntington's Disease

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by an increase in the number of polyglutamine residues in the huntingtin (Htt) protein. With the identification of the underlying basis of HD, therapies are being developed that reduce expression of the causative mutant Htt. RNA interference (RNAi) that seeks to selectively reduce the expression of such disease-causing agents is emerging as a potential therapeutic strategy for this and similar disorders. This study examines the merits of administering a recombinant adeno-associated viral (AAV) vector designed to deliver small interfering RNA (siRNA) that targets the degradation of the Htt transcript. The aim was to lower Htt levels and to correct the behavioral, biochemical, and neuropathological deficits shown to be associated with the YAC128 mouse model of HD. Our data demonstrate that AAV-mediated RNAi is effective at transducing greater than 80% of the cells in the striatum and partially reducing the levels (~40%) of both wild-type and mutant Htt in this region. Concomitant with these reductions are significant improvements in behavioral deficits, reduction of striatal Htt aggregates, and partial correction of the aberrant striatal transcriptional profile observed in YAC128 mice. Importantly, a partial reduction of both the mutant and wild-type Htt levels is not associated with any notable overt neurotoxicity. Collectively, these results support the continued development of AAV-mediated RNAi as a therapeutic strategy for HD.

P.19.2 Targeting the Huntingtin gene with splice-switching AOs: An unusual result

Huntington's disease is a late-onset neuromuscular disorder caused by the abnormal expansion of a trinucleotide repeat (CAG) that codes for glutamine in exon 1 of the Huntingtin gene (HTT). Both the mutated protein and RNA transcript induce a toxic effect, altering downstream interactions and causing neural degeneration and a decline in cognitive and muscle function. Antisense oligonucleotides have been used to knockdown the expression of mutant HTT, either by inducing RNase H or blocking translation. However, most of these approaches target the repeat itself, which could lead to non-specific effects. This study used 2′-O-methyl AOs, which do not induce RNase H activity and are often used to alter exon selection to target sequences surrounding the repeat region. The hypothesis was that the presence of the expanded repeat is likely to alter the way splicing factors interact, and targeting an AO to exon 1 could induce the removal of the exon in a mutation-specific manner, leaving the wild type allele intact while knocking down the expanded mutant allele. Fourteen oligomers targeted to exon 1 were tested initially, and three of these altered pre-mRNA processing, as RT-PCR amplification demonstrated the presence of smaller products that did not follow exon boundaries. This phenomenon, whereby nucleotides from the middle of an exon are removed, has not been reported elsewhere. In silico analysis of potential splice sites indicates there are no potential splice sites near the excluded region. Further studies are currently being conducted to determine possible mechanisms of exon excision, and the implications for AO targeting of GC rich sequences.

Disassociation of Histone Deacetylase-3 from Normal Huntingtin Underlies Mutant Huntingtin Neurotoxicity

Huntington's disease (HD) is caused by a polyglutamine expansion within the huntingtin (Htt) protein. Both loss of function of normal Htt and gain of a toxic function by the polyglutamine-expanded mutant Htt protein have been proposed to be responsible for HD, although the molecular mechanisms involved are unclear. We show that Htt is a neuroprotective protein in both HD-related and unrelated model systems. Neuroprotection by Htt is mediated by its sequestration of histone deacetylase-3 (HDAC3), a protein known to promote neuronal death. In contrast to the normal Htt, mutant Htt interacts poorly with HDAC3. However, expression of mutant Htt liberates HDAC3 from Htt, thus de-repressing its neurotoxic activity. Indeed, mutant Htt neurotoxicity is inhibited by the knockdown of HDAC3 and markedly reduced in HDAC3-deficient neurons. A reduction in Htt-HDAC3 interaction is also seen in neurons exposed to other apoptotic stimuli and in the striatum of R6/2 HD mice. Our results suggest that the robust interaction between Htt and HDAC3 along with the ability of mutant Htt to disrupt this association while not itself interacting with HDAC3 provides an explanation for both the loss-of-function and gain-of-toxic-function mechanisms proposed for HD. Moreover, our results identify HDAC3 as an essential player in mutant Htt-induced neurodegeneration.

MiR-196a Ameliorates Phenotypes of Huntington Disease in Cell, Transgenic Mouse, and Induced Pluripotent Stem Cell Models

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder characterized by dysregulation of various genes. Recently, microRNAs (miRNAs) have been reported to be involved in this dysregulation, suggesting that manipulation of appropriate miRNA regulation may have a therapeutic benefit. Here, we report the beneficial effects of miR-196a (miR196a) on HD in cell, transgenic mouse models, and human induced pluripotent stem cells derived from one individual with HD (HD-iPSCs). In the in vitro results, a reduction of mutant HTT and pathological aggregates, accompanying the overexpression of miR-196a, was observed in HD models of human embryonic kidney cells and mouse neuroblastoma cells. In the in vivo model, HD transgenic mice overexpressing miR-196a revealed the suppression of mutant HTT in the brain and also showed improvements in neuropathological progression, such as decreases of nuclear, intranuclear, and neuropil aggregates and late-stage behavioral phenotypes. Most importantly, miR-196a also decreased HTT expression and pathological aggregates when HD-iPSCs were differentiated into the neuronal stage. Mechanisms of miR-196a in HD might be through the alteration of ubiquitin-proteasome systems, gliosis, cAMP response element-binding protein pathway, and several neuronal regulatory pathways in vivo. Taken together, these results show that manipulating miR-196a provides beneficial effects in HD, suggesting the potential therapeutical role of miR-196a in HD.

Evidence for dynamic and multiple roles for huntingtin in Ciona intestinalis

Although mutations in the huntingtin gene (HTT) due to poly-Q expansion cause neuropathology in humans (Huntington’s disease; HD), the normal function(s) of the gene and its protein (HTT) remain obscure. With new information from recently sequenced invertebrate genomes, the study of new animal models opens the possibility of a better understanding of HTT function and its evolution. To these ends, we studied huntingtin expression pattern and dynamics in the invertebrate chordate Ciona intestinalis. Ciona huntingtin (Ci-HTT) shows a biphasic expression pattern during larval development and prior to metamorphosis. A single form of huntingtin protein is present until the early larval stages, at which time two different mass proteins become evident in the metamorphically competent larva. An antibody against Ci-HTT labeled 50 cells in the trunk mesenchyme regions in pre-hatching and hatched larvae and probably represents the distribution of the light form of the protein. Dual labeling with anti-Ci-HTT and anti-aldoketoreductase confirmed the presence of Ci-HTT in mesenchyme cells. Suppression of Ci-HTT RNA by a morpholino oligonucleotide reduced the number and apparent mobility of Ci-HTT positive cells. In Ciona, HTT expression has a dynamic temporal and spatial expression pattern that in ontogeny precedes metamorphosis. Although our results may reflect a derived function for the protein in pre- and post-metamorphic events in Ciona, we also note that as in vertebrates, there is evidence for multiple differential temporal expression, indicating that this protein probably has multiple roles in ontogeny and cell migration.

Non-invasive delivery of small interfering ribonucleic acid for reduction of Huntingtin expression in the brain is achieved using focused ultrasound to disrupt the blood-brain barrier

Huntington’s disease is caused by a mutation in the Huntingtin (Htt) gene, which leads to neuronal dysfunction and cell death. Silencing of the Htt gene can halt or reverse the progression of the disease indicating that RNA interference is the most effective strategy for disease treatment. However, small interfering RNA (siRNA) does not cross the blood-brain barrier and therefore delivery to the brain is limited. Here, we demonstrate that focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48 hours following treatment with siRNA, the right (treated) and left (control) striatum was dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients.

Huntingtin and HAP1 Regulate Microtubule Motors during Autophagosome Transport

Autophagy is a critical homeostatic process whereby autophagosomes engulf and degrade cellular cargo. Live cell imaging assays in neurons recently demonstrated autophagosome formation is restricted to the axon tip, followed by robust retrograde transport of the autophagosome towards the cell body. While >75% of autophagosome motility is directed towards microtubule minus ends, both the minus end-directed motor dynein and the plus end-directed motor kinesin colocalize to autophagosomes in neurons and copurify with isolated autophagosomes. Thus, it still remains unclear what mechanisms promote autophagosome transport, activating the retrograde motor dynein while inactivating the anterograde motor kinesin. One key candidate for regulating autophagosome transport is huntingtin (htt), which directly binds to dynein and binds to kinesin and dynactin through the adaptor HAP1 (huntingtin associated protein 1). Using live cell imaging in primary neurons, we found that depleting endogenous htt disrupted the retrograde motility of autophagosomes resulting in shorter run lengths and slower velocities. While these defects could be rescued by expression of full-length htt, deletion constructs of htt that could not bind either dynein or HAP1 were unable to rescue the defects in autophagosome motility upon depletion of endogenous htt. To further test HAP1's role in regulating autophagosome transport, we depleted endogenous HAp1 and observed a similar disruption in the retrograde motility of autophagosomes. Finally, we found that autophagosome transport is disrupted in a neuronal model of Huntington's disease, a genetic neurodegenerative disorder characterized by polyglutamine expansions in htt. Our results suggest a model where htt regulates autophagosome transport by binding to dynein and enhancing motor processivity, as well as to HAP1 whose interactions with kinesin and dynactin may further promote retrograde autophagosome transport. Further, out data suggest that misregulation of this transport may contribute to the pathogenesis of Huntington's disease.

Defective mitochondrial disulfide relay system, altered mitochondrial morphology and function in Huntington's disease

A number of studies have been conducted that link mitochondrial dysfunction (MD) to Huntington's disease (HD); however, contradicting results had resulted in a lack of a clear mechanism that links expression of mutant Huntingtin protein and MD. Mouse homozygous (HM) and heterozygous (HT) mutant striatal cells with two or one allele encoding for a mutant huntingtin protein with 111 polyGln repeats showed a significant impairment of the mitochondrial disulfide relay system (MDRS). This system (consisting of two proteins, Gfer and Mia40) is involved in the mitochondrial import of Cys-rich proteins. The Gfer-to-Mia40 ratio was significantly altered in HM cells compared with controls, along with the expression of mitochondrial proteins considered substrates of the MDRS. In progenitors and differentiated neuron-like HM cells, impairment of MDRS were accompanied by deficient oxidative phosphorylation, Complex I, IV and V activities, decreased mtDNA copy number and transcripts, accumulation of mtDNA deletions and changes in mitochondrial morphology, consistent with other MDRS-deficient biological models, thus providing a framework for the energy deficits observed in this HD model. The majority (>90%) of the mitochondrial outcomes exhibited a gene-dose dependency with the expression of mutant Htt. Finally, decreases in the mtDNA copy number, along with the accumulation of mtDNA deletions, provide a mechanism for the progressive neurodegeneration observed in HD patients.

Glutathione redox cycle dysregulation in Huntington’s disease knock-in striatal cells

Huntington’s disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and γ-glutamyl transpeptidase (γ–GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to counterbalance increased ROS formation and emergent apoptotic features in HD striatal cells.

C09 Characterisation of a rat model of Huntington's disease based on targeted expression of a mutant huntingtin fragment in the forebrain using an adeno-associated viral vector

Background Huntington9s disease (HD) is characterised by motor symptoms, cognitive decline and psychiatric disturbances. The neuropathology is widespread but the striatum and the cerebral cortex are among the most severely affected areas. As mutant huntingtin (htt) is ubiquitously expressed, it has been difficult to establish structure-to-function relationships for the development of the different clinical manifestations. Aims Our aim was to develop a new rat model of HD with targeted forebrain expression of htt. Methods We have injected recombinant adeno-associated viral vectors of serotype 5 (rAAV5) expressing an 853 amino acid fragment of either wildtype (htt853-18Q) or mutant (htt853-79Q) htt, or green fluorescent protein (GFP) in the dorsal striatum of neonatal rats. Adult rats were assessed for neuropathology, body weight, anxiety–like behaviour and motor activity at 2 and 6 months post-injection. Results/Outcomes Neuropathological assessment at 6 months showed long-term expression of GFP in the cerebral cortex and the dorsal striatum. In htt853-79Q injected brains we found ubiquitinated htt inclusions mainly in the cortical region. No gliosis or microglia activation was detected and stereological quantifications of NeuN- and DARPP-32 positive cells revealed no cell loss after expression of htt853-79Q. Interestingly, the same vector injected into the hypothalamus led to rapid formation of htt inclusions and orexin loss. No differences in the general motor activity or body weight were observed after targeted forebrain expression of the mutant htt fragment. However, overexpression of htt853-18Q led to significantly less anxiety-like behaviour. Conclusions This AAV-vector mediated model of HD demonstrated widespread forebrain expression of mutant htt and could be an interesting tool to investigate specific effects of htt in different brain regions.

A11 Induced pluripotent stem cells for basic and translational research on HD

<h3>Background</h3> The expression of mutant HTT leads to many cellular alterations, including abnormal vesicle recycling, loss of signalling by brain-derived neurotrophic factor, excitotoxicity, perturbation of Ca²⁺ signalling, decreases in intracellular ATP, alterations of gene transcription, inhibition of protein clearance pathways, mitochondrial and metabolic disturbances, and ultimately cell death. While robust mammalian systems have been developed to model disease and extensive mechanistic insights have emerged, significant differences between rodent and human cells and between non-neuronal cells and neurons limit the utility of these models for accurately representing human disease. Human pluripotent stem cells can generate highly specified cell populations, including DARPP32-positive MSNs of the striatum, and provide a method for modelling HD in human neurons carrying the mutation. As it is caused by one single gene, HD is an ideal disorder for exploring the utility of modelling disease in induced pluripotent stem cells (iPSCs) through reprogramming adult cells from HD patients with known patterns of disease onset and duration. <h3>Aims</h3> Generate iPSC lines from HD patients and controls and identify CAG-repeat expansion associated phenotypes. <h3>Methods/techniques</h3> Through the efforts of an international consortium effort, 14 lines were generated, differentiated into neuronal populations and assessed for CAG-repeat dependent outcome measures. <h3>Results/outcomes</h3> HD iPSC lines have reproducible CAG expansion–associated phenotypes upon differentiation, including CAG expansion-associated changes in gene expression patterns and alterations in electrophysiology, metabolism, cell adhesion, and ultimately an increased risk of cell death. While the lines with the longest repeats (HD180) showed a phenotype across all assays, those with shorter repeats (HD60) showed phenotypes in a specific sub set of assays. The most sensitive assay for establishing repeat dependent effects was found to be calcium responses to stress. <h3>Conclusions</h3> This HD iPSC collection represents a unique and well-characterised resource to elucidate disease mechanisms in HD and provides a novel human stem cell platform for screening new candidate therapeutics. <h3>Funding</h3> NIH, CHDI, CIRM.

B06 Genotoxic stress in fibroblasts and mesenchymal stem cells isolated from miniature pigs transgenic for n-terminal part of mutated human huntingtin

Background Huntington9s disease (HD) is a dominant inherited disorder caused by mutation in huntingtin (htt) protein that causes massive neural cell death leading to neurodegeneration. Mutated huntingtin (mt htt) induces a cascade of events that stimulates DNA breaks and the activation of DNA damage response pathway. This genotoxic stress is followed by activation of the MDC1 and H2AX proteins, in the early stages of HD pathogenesis. Expression of htt occurs in cells of neuronal and non-neuronal origin. Aims The aim of our research is to study genetoxic stress of mt htt on fibroblasts and mesenchymal stem cells (MSCs) of our miniature pig model for HD. Methods Currently, we have three generations of transgenic (tg) minipigs for N-terminal part of the human mt htt (548 aa, 124 Q). Each litter presents wild type (wt) and tg siblings at an approximate ratio 1:1 with the same genetic background. DNA damage was examined on fibroblasts and MSCs isolated from tg and wt minipigs using confocal microscopy, immunocytochemistry, Western blot analysis and the dynamic of tg and wt cell proliferation was tested by life cell imaging. Results The significant increase of MDC1 and γH2AX proteins, indicating DNA damage, was proved by immunocytochemistry and also Western blot analysis. Proliferation tests could not clearly establish the genotoxic stress of tg fibroblasts and MSCs. Conclusions We will continue in our studies to observe changes in genotoxic stress during the lifespan of our minipigs. HD tg miniature pig represents a perspective large animal model for the research of molecular mechanisms of mt htt.

F01 Establishment of novel assays for quantification of full-length wild-type and mutant huntingtin by using the alphalisa technology

<h3>Background</h3> Huntington9s disease (HD) is caused by a CAG repeat expansion in the huntingtin (htt) gene and there is no cure available. Currently, there are several animal models that recapitulate the disease phenotype and knockdown of mutant htt in some of these models have exhibited beneficial therapeutic effects. However, there are limited tools to quantify different forms of soluble full-length htt in biological samples in order to monitor the efficacy of the therapeutic molecules. <h3>Aim</h3> We aimed to establish bioassays that can detect and selectively quantify the wild-type (WT) and mutant forms of htt in biological samples. <h3>Methods</h3> Two-step no wash bioassays were developed to detect soluble forms of full-length htt by using the AlphaLISA® technology. In each assay, a pair of antibody was used to capture either total or mutant htt. Assay performance was tested in HEK293 cells, WT and BACHD brain homogenates. For selective quantification of the WT htt, brain samples from BACHD mice were incubated with antibody-conjugated magnetic beads to deplete mutant htt. <h3>Results</h3> The AlphaLISA® platform was efficient at detecting endogenous and overexpressed forms of soluble htt in HEK293 cells and in brain homogenates. The mutant htt assay showed a signal increase proportional to the length of the glutamine repeat. Total and mutant htt assays revealed differential htt expression in various brain regions (cortex, hippocampus, thalamus and cerebellum) of WT and BACHD mouse models. In addition, our results showed that total htt levels in BACHD brain is only about 40%–50% higher than WT mice, suggesting that this mouse model does not exhibit overt overexpression of the protein. <h3>Conclusions</h3> Htt AlphaLISA assays demonstrated robust performance, high sensitivity and broad dynamic range. Hence, these assays can be used as a powerful tool to quantify soluble htt variants in experimental studies using both cellular and tissue samples.

Focused ultrasound for targeted delivery of siRNA and efficient knockdown of Htt expression

RNA interference is a promising strategy for the treatment of Huntington's disease (HD) as it can specifically decrease the expression of the mutant Huntingtin protein (Htt). However, siRNA does not cross the blood–brain barrier and therefore delivery to the brain is limited to direct CNS delivery. Non-invasive delivery of siRNA through the blood–brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients. Focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48h following treatment with siRNA, the right (treated) and the left (control) striatum were dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain.

Choosing and usingDrosophilamodels to characterize modifiers of Huntington's disease

HD (Huntington's disease) is a fatal inherited gain-of-function disorder caused by a polyQ (polyglutamine) expansion in the htt (huntingtin protein). Expression of mutant htt in model organisms is sufficient to recapitulate many of the cellular defects found in HD patients. Many groups have independently developed Drosophila models of HD, taking advantage of its rapid life cycle, carefully annotated genome and well-established molecular toolkits. Furthermore, unlike simpler models, Drosophila have a complex nervous system, displaying a range of carefully co-ordinated behaviours which offer an exquisitely sensitive readout of neuronal disruption. Measuring HD-associated changes in behaviour in Drosophila therefore offers a window into the earliest stages of HD, when therapeutic interventions might be particularly effective. The present review describes a number of recently developed Drosophila models of HD and offers practical guidance on the advantages and disadvantages of various experimental approaches that can be used to screen these models for modifiers of mutant htt-mediated toxicity.

Adenovirus vector‐based in vitro neuronal cell model for Huntington's disease with human disease‐like differential aggregation and degeneration

Neuronal degeneration, in particular in the striatum, and the formation of nuclear and cytoplasmic inclusions are characteristics of Huntington's disease (HD) as a result of the expansion of a polyglutamine tract located close to the N-terminus of huntingtin (htt). Because of the large (10-kb) size of the htt cDNA, expression of full-length htt in primary neurons has proved difficult in the past. We generated a new chronic in vitro model that is based on high-capacity adenovirus vector-mediated transduction of primary murine striatal and cortical neurons. Because the vector has a large capacity for transport of foreign DNA, it was possible to quantitatively express in these primary cells normal and mutant full-length htt (designed as fusion proteins with enhanced green fluorescent protein) in addition to its truncated versions. Pathological changes caused by mutant htt were characterized. The model mimicked several features observed in HD patients: prominent nuclear inclusions in cortical but not in striatal neurons, preferential neuronal degeneration of striatal neurons and neurofilament fragmentation in this cell type. Compared with expressed truncated mutant htt, the expression of full-length mutant htt in neurons resulted in a much slower appearance of pathological changes. Different from cortical neurons, the vast majority of nuclei in striatal cells contained only diffusely distributed N-terminal htt fragments. Cytoplasmic inclusions in both cell types contained full-length mutant htt. This model and the adenovirus vectors used will be valuable for studying the function of htt and the pathogenesis of HD at molecular and cellular levels in different neuronal cell types.

Trans-(−)-ε-Viniferin Increases Mitochondrial Sirtuin 3 (SIRT3), Activates AMP-activated Protein Kinase (AMPK), and Protects Cells in Models of Huntington Disease

Huntington disease (HD) is an inherited neurodegenerative disorder caused by an abnormal polyglutamine expansion in the protein Huntingtin (Htt). Currently, no cure is available for HD. The mechanisms by which mutant Htt causes neuronal dysfunction and degeneration remain to be fully elucidated. Nevertheless, mitochondrial dysfunction has been suggested as a key event mediating mutant Htt-induced neurotoxicity because neurons are energy-demanding and particularly susceptible to energy deficits and oxidative stress. SIRT3, a member of sirtuin family, is localized to mitochondria and has been implicated in energy metabolism. Notably, we found that cells expressing mutant Htt displayed reduced SIRT3 levels. trans-(-)-ε-Viniferin (viniferin), a natural product among our 22 collected naturally occurring and semisynthetic stilbenic compounds, significantly attenuated mutant Htt-induced depletion of SIRT3 and protected cells from mutant Htt. We demonstrate that viniferin decreases levels of reactive oxygen species and prevents loss of mitochondrial membrane potential in cells expressing mutant Htt. Expression of mutant Htt results in decreased deacetylase activity of SIRT3 and further leads to reduction in cellular NAD(+) levels and mitochondrial biogenesis in cells. Viniferin activates AMP-activated kinase and enhances mitochondrial biogenesis. Knockdown of SIRT3 significantly inhibited viniferin-mediated AMP-activated kinase activation and diminished the neuroprotective effects of viniferin, suggesting that SIRT3 mediates the neuroprotection of viniferin. In conclusion, we establish a novel role for mitochondrial SIRT3 in HD pathogenesis and discovered a natural product that has potent neuroprotection in HD models. Our results suggest that increasing mitochondrial SIRT3 might be considered as a new therapeutic approach to counteract HD, as well as other neurodegenerative diseases with similar mechanisms.

Characterization of a rat model of Huntington’s disease based on targeted expression of mutant huntingtin in the forebrain using adeno‐associated viral vectors

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (htt) gene. Neuropathology is most severe in the striatum and cerebral cortex. As mutant htt is ubiquitously expressed, it has not been possible to establish clear structure-to-function relationships for the clinical aspects. In the present study, we have injected recombinant adeno-associated viral vectors of serotype 5 (rAAV5) expressing an 853-amino-acid fragment of htt with either 79 (mutant) or 18 (wild-type) glutamines (Q) in the dorsal striatum of neonatal rats to achieve expression of htt in the forebrain. Rats were followed for 6 months and compared with control rats. Neuropathological assessment showed long-term expression of the green fluorescent protein (GFP) transgene (used as a marker protein) and accumulation of htt inclusions in the cerebral cortex with the rAAV5-htt-79Q vectors. We estimated that around 10% of NeuN-positive cells in the cerebral cortex and 2% of DARPP-32 neurons in the striatum were targeted with the GFP-expressing vector. Formation of intracellular htt inclusions was not associated with neuronal loss, gliosis or microglia activation and did not lead to altered motor activity or changes in body weight. However, the same mutant htt vector caused orexin loss in the hypothalamus - another area known to be affected in HD. In conclusion, our results demonstrate that widespread forebrain expression of mutant htt can be achieved using rAAV5-vectors and suggest that this technique can be further explored to study region-specific effects of mutant htt or other disease-causing genes in the brain.

Rab11 rescues synaptic dysfunction and behavioural deficits in a Drosophila model of Huntington's disease

Synapse abnormalities in Huntington's disease (HD) patients can precede clinical diagnosis and neuron loss by decades. The polyglutamine expansion in the huntingtin (htt) protein that underlies this disorder leads to perturbations in many cellular pathways, including the disruption of Rab11-dependent endosomal recycling. Impairment of the small GTPase Rab11 leads to the defective formation of vesicles in HD models and may thus contribute to the early stages of the synaptic dysfunction in this disorder. Here, we employ transgenic Drosophila melanogaster models of HD to investigate anomalies at the synapse and the role of Rab11 in this pathology. We find that the expression of mutant htt in the larval neuromuscular junction decreases the presynaptic vesicle size, reduces quantal amplitudes and evoked synaptic transmission and alters larval crawling behaviour. Furthermore, these indicators of early synaptic dysfunction are reversed by the overexpression of Rab11. This work highlights a potential novel HD therapeutic strategy for early intervention, prior to neuronal loss and clinical manifestation of disease.